Solubility of Foreign Molecules in Stratum Corneum Brick and Mortar Structure

The barrier function of the skin is mainly assured by its outermost layer, stratum corneum (SC). One key aspect in predicting dermal drug delivery and in safety assessment of skin exposure to chemicals is the need to determine the amount of chemical that is taken up into the SC. We here present a strategy that allows for direct measures of the amount of various solid chemicals that can be dissolved in the SC in any environmental relative humidity (RH). A main advantage of the presented method is that it distinguishes between molecules that are dissolved within the SC and molecules that are not dissolved but might be present at, for example, the skin surface. In addition, the method allows for studies of uptake of hydrophobic chemicals without the need to use organic solvents. The strategy relies on the differences in the molecular properties of the added molecules in the dissolved and the excess states, employing detection methods that act as a dynamic filter to spot only one of the fractions, either the dissolved molecules or the excess solid molecules. By measuring the solubility in SC and delipidized SC at the same RHs, the same method can be used to estimate the distribution of the added chemical between the extracellular lipids and corneocytes at different hydration conditions. The solubility in porcine SC is shown to vary with hydration, which has implications for the molecular uptake and transport across the skin. The findings highlight the importance of assessing the chemical uptake at hydration conditions relevant to the specific applications. The methodology presented in this study can also be generalized to study the solubility and partitioning of chemicals in other heterogeneous materials with complex composition and structure.

. (A) WAXS profiles of 2-MR and of SC sheets with different amount of 2-MR at RH=97% (D2O) and at 32 C. The sample was prepared and equilibrated in the same way as pulverized SC samples. The data show that the saturated mass fraction in SC FSC,sat at 97% RH of 2-MR in SC sheets is 40-42.5 wt% which is rather similar to the data obtained for pulverized SC (Table 1). Scattering peaks from keratin interchain distance (K) and lipid chain packing (L) are also labeled. * indicates peaks from Kapton (see Fig. S1B). (B) WAXS profiles of only Kapton and of pulverized SC with 3 wt% caffeine at 97% RH. The amount of the added chemicals in wt% refers to their mass fraction in SC calculated as mAC-SC/(mAC-SC + mSC)⋅100%. mAC-SC and mSC are the weight of the added chemicals AC in SC and the dry weight of SC, respectively.

Fig. S2
. WAXS profiles recorded during different time points after mixing SC and pure 2-methyl resorcinol (with a proportion corresponding to 2 wt% 2-methyl resorcinol in samples of SC and 2-methyl resorcinol) that had been equilibrated separately at RH=93% (D2O) and at 32 C. The earliest measured time point is limited by the instrument operation. After 9.5 minutes after mixing, we could not detect any signs of solid 2-MR in the sample, indicating that the dissolution of solid 2-MR into SC at RH=93% is faster than 9.5 minutes. The time resolution can be improved by using synchrotron WAXS instruments with higher flux together with experimental set-ups where the time gap between mixing and starting the measurement is shorter. The amount of the added chemicals in wt% refers to their mass fraction in SC calculated as mAC-SC/(mAC-SC + mSC)⋅100%.

Supplementary Section 1. Measuring solubility of caffeine and 2-MR in H2O
The solubility of the added chemicals in H2O was measured on samples of saturated chemical in H2O equilibrated with excess amount of the solid chemical at 32 C for 2 weeks. From the area ratio of the peaks in 1 H MAS NMR spectra of the saturated solutions at 32 C (Fig. S6), one can estimate the molar ratios of the chemical and H2O of 407 mol H2O per mol caffeine and 14.6 mol H2O per mol 2-MR, corresponding to 26 g caffeine per 1000 g caffeine and H2O and 321 g 2-MR per 1000 g 2-MR and H2O. If we assume the density of the saturated solution of the chemicals is 1000 g/L, we can obtain the solubility in water of caffeine of 26 g/L and of 2-MR of 321 g/L.

Supplementary Section 2. Details on calculations of solubility of added chemicals in SC at fully hydrated condition from previous studies 1, 2
Most of previous studies on solubility in SC report on the partition coefficients of added chemicals between SC and an excess aqueous solutions. 1,2 We need to recalculate the maximum amount of the added chemical dissolved within SC from the partition coefficients in order to make direct quantitative comparisons with the data in Table 1. The partition coefficient SC/W of chemicals between SC and an aqueous solution (W) is defined as the ratio between the solubility in SC and the solubility in water, expressed in mass of the chemical per volume of SC or the aqueous solution. From the literature data on measured partition coefficients and solubility of the chemical in water, we can calculate the maximum amount of the added chemical dissolved within SC per volume of SC. This can then be recalculated to SSC by knowing the density of fully hydrated SC.

SC/W is defined as
where mAC-SC and mAC-W refer to the weight of the added chemical (AC) in SC and in the aqueous solution (W), respectively. fully hydrated SC and W refer to the volume of fully hydrated SC and volume of the aqueous solution. In saturated condition of chemical, AC−W W corresponds to the solubility of chemical in water SW (mg/mL).
The volume of hydrated SC has been proposed to be 3.518 mL per 1 g dry human SC 3 or fully hydrated SC = 3.518 ⋅ 10 −3 in which the units of fully hydrated SC and SC are mL and mg, respectively.
From Eq. S1 and S2, we then obtain the following expression: Using the solubility in H2O water of caffeine (26 mg/mL, Table 1) and SC/W value for caffeine obtained from pig SC 1 or human SC 2 in previous studies, we can obtain FSC,sat values of caffeine in fully hydrated condition according to Eq. S5 and the values are shown in Table  S1. The value of SC/W can also be estimated from log PO/W (reviewed in 4 ) but we herein use experimental values for this estimation. Using a water content of 2.75 g H2O water per 1 g dry SC (or 3.05 g D2O water per 1 g dry SC) for fully hydrated human SC, 3 the solubility of caffeine in SC at full hydration then can be estimated in Table S1. It is noted that the volume of hydrated SC and the water content per 1 g dry SC used in the estimation are of human SC. Table S1. Estimated solubility of caffeine in SC at full hydration from the partition coefficients KSC/W obtained from previous studies. 1,2 The solubility in SC is defined as SSC=[mAC-SC-sat/(mAC-SC-sat+ mSC+ mW-SC)⋅100%] (wt%) and the saturated mass fraction in SC is FSC,sat=mAC-SC-sat/(mAC-SC-sat+ mSC)⋅100% (wt%). mAC-SC-sat and mW-SC are the weights of the added chemicals and water in SC, respectively at the saturation condition of the added chemical. mSC refers to the dry weight of SC. Solubility of the chemicals in H2O water SW (mg/mL) is also shown.